The discovery of circulating cell-free DNA (ccfDNA) in the human circulatory system has led to intensive research on its use in various clinical fields. CcfDNA was discovered in 1948 by Mandel and Metais1 although at the time, it did not attract much curiosity. Thirty to 40 years later, however, the interest of ccfDNA was demonstrated by several groups: Leon et al.2 found that ccfDNA concentration was significantly increased in cancer subjects and Stroun et al.3 described a proportion of ccfDNA that was tumor derived and carried its molecular characteristics, thus leading to the concept of a “liquid biopsy”. Additionally, ccfDNA fragmentation has grown in interest in terms of diagnosis since the revelation of significant differences between cancer subjects and healthy subjects4 (Stroun U.S. Pat. No. 5,952,170). Therefore, ccfDNA analysis could provide diagnostic, pronostic, and theranostic information5. Several researchers are intensively developing techniques that allow detection and characterization of genetic and epigenetic alterations of tumor cells using ccfDNA analysis in the plasma/serum of cancer subjects. Such techniques could revolutionize the management care of cancer subjects through the detection of mutations leading to resistance to targeted therapies, personalized therapeutic monitoring and non-invasive follow-up of the disease. ccfDNA analysis is currently used in prenatal diagnosis practice6 and a promising analysis in other clinical fields, such as autoimmune diseases, trauma, sepsis, or myocardial infarction7.
Despite intensive research, few ccfDNA-based tests have been translated to clinical practice. Several techniques are under development to detect and characterize ccfDNA in cancer subjects including restriction fragment length polymorphism, direct sequencing, high-resolution melting analysis, digital PCR, cold PCR, and other techniques usually used for tumor-tissue analysis. Nevertheless, ccfDNA concentration has not yet been validated as a cancer biomarker as the literature reveals conflicting data: plasma ccfDNA concentrations in cancer subjects range from a few ng/ml to several thousand ng/ml, which overlaps with the concentration range for healthy individuals5. Furthermore, the estimation of ccfDNA fragmentation in cancer subjects has been found to be lower, equivalent, or higher than in control subjects. These discrepancies may be explained by the lack of fundamental knowledge about ccfDNA. Indeed, the cognitive aspects of ccfDNA are still not identified and elucidated: The respective contributions of different potential release mechanisms of ccfDNA (apoptosis, necrosis, phagocytosis, extracellular DNA traps, active release . . . ) are not clearly identified. Similarly, structures of ccfDNA are not yet clearly defined (part of chromatin, nucleosomes, nucleoprotein complex, exosomes, apoptotic bodies . . . )8.
Total concentration of circulating DNA was envisaged for long time as a potential biomarker for cancer but cfDNA concentration values from healthy and cancer individuals were partly found overlapping precluding its development as clinical use9. With recent methods and specific PCR system design, statistical discrimination was recently showed between healthy subjects and cancer subjects10. Moreover, we revealed that circulating tumor DNA is highly fragmented in comparison of cfDNA from healthy subjects (U520130224740,11). Targeting short sequences lead to find that up to 50% of total ccfDNA could be derived from the tumor12 breaking the previous literature statement describing that tumor-derived ccfDNA was a tiny portion of total ccfDNA.
Nevertheless, structural and size characteristics of ccfDNA are still poorly characterized in the literature while it could contribute to improve cognitive knowledge on ccfDNA and to design accurate and specific analytical processes. In addition to the current vigourous research on nuclear ccfDNA (CnDNA) analysis, mitochondrial ccfDNA (CnDNA) analysis is emerging as a very attractive study field.
Mitochondrial DNA (mtDNA) is composed of a circular DNA of 16,000 bp inserting to 37 genes, coding for two rRNAs, 22 tRNAs and 14 polypeptides (above presented, one encoding both ribosomal 16S and humanin). They are 5 to 10 copies of this circular DNA per mitochondria, and exist only in eukaryotes in all types of cells except of red cells; each cells containing 1000 to 3000 mitochondria upon cell types. Thus, there are hundreds to thousands copies of mtDNA, number variation being function upon environmental conditions (such as hypoxia or steroid hormone stimulation)13. mtDNA corresponds to about 1% of the total cellular DNA. Since it is derived from bacterial DNA it contains numerous unmethylated CpG dinucleotides14. Because of lack of protection by histones and efficient DNA repair mechanisms, it is sensitive to genotoxic and oxidative stress15. There is 10 to 200-fold higher mutation rate than nuclear DNA. During tumorogenesis, mitochondrial DNA is subjected to many mutations in much higher proportion than nuclear DNA due to its lack of protection by histones and lack of DNA repair mechanisms. Moreover, this polyploid genome is subjected to copy number variation during carcinogenesis. These specific alterations could be easily determined from mitochondrial ccfDNA since mtDNA copies are present in higher quantity (hundred to thousand copies by cell) than nuclear DNA13. Some studies have been published on the clinical significance of mitochondrial ccfDNA concentration and integrity in cancer subjects16. At this time, published data are discordant and it is impossible to draw any conclusion. The lack of preanalytical and analytical SOP could explain in part this discordance.
Little is known about the structural properties of mitochondrial ccfDNA (CmDNA). We can hypothesize that they are different of nuclear ccfDNA (CnDNA) since mDNA is not protected by histones and a part of nuclear ccfDNA is made of nucleosomes. It has been shown that Neutrophil released mDNA in a ROS manner dependant to form the Neutrophil Extracellular Traps (NET)17. Similarly, Eosinophil release mtDNA to form the extracellular traps (EET) contributing to antibacterial defense18.
Biologically and physiologically, it has been demonstrated that mitochondrial ccfDNA was a DAMP14. Its specific unmethylated CpG pattern and its similar characteristics with bacterial DNA is recognized by TLR9 of immune cells and led to inflammatory response via p3819. This point is crucial since little is known about biological properties of ccfDNA and such a discovery could lead to therapeutic agents directed against ccfDNA. For all these reasons, mitochondrial ccfDNA is very promising and further studies on this particular DNA need to be achieved. It is poorly characterized20 and few results have been reported as compared to nuclear ccfDNA.